Vitamin E attenuates crystal formation in rat kidneys: Roles of renal tubular cell death and crystallization inhibitors  H.-S. Huang, J. Chen, C.-F. Chen, M.-C. Ma  Kidney International  Volume 70, Issue 4, Pages 699-710 (August 2006) DOI: 10.1038/sj.ki.5001651 Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 1 Representative micrographs of renal CaOx crystal deposition photographed using a polarized microscopy. Left panels: Effect of 0.75% EG in the drinking water; (a) untreated control group, (b–d) rats treated with EG for (b) 7, (c) 21, or (d) 42 days. Right panels: Effect of vitamin E supplementation; (e) E-treated control group, (f–h) rats treated with EG+E for (f) 7, (g) 21, or (h) 42 days. Reduced from × 100. The white arrows indicate CaOx crystal deposition. (i) Statistical result for Tiselius Risk Index calculation. *P<0.05 compared to the corresponding control group. Kidney International 2006 70, 699-710DOI: (10.1038/sj.ki.5001651) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 2 Representative micrographs of CaOx crystals in urine sediments photographed using a polarized microscopy. Left two bars: Effect of 0.75% EG on (a) untreated controls or (b–d) rats treated with EG for (b) 7, (c) 21, or (d) 42 days. Right two bars: Effect of vitamin E supplementation on (e) E-treated controls or (f–h) EG rats treated with vitamin E for (f) 7, (g) 21, or (h) 42 days. Reduced from × 100. (i) Statistical result for the dry weight of urine sediments. *P<0.05 compared to the corresponding control group. Kidney International 2006 70, 699-710DOI: (10.1038/sj.ki.5001651) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 3 Effect of vitamin E on tubular cell damage and urinary lipid peroxide excretion in the EG-treated kidneys. (a) Temporal changes in urine enzymuria (N-acetyl-β-glucosaminidase) and LDH levels in renal tissues and (b) serum levels of vitamin E and urinary excretion of malondialdehyde and thiobarbituric acid-reactive substances in the age-matched untreated control and EG groups and the E-treated control and EG+E groups. Kidney International 2006 70, 699-710DOI: (10.1038/sj.ki.5001651) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 4 Representative micrographs of renal sections stained for αGST. Left panels: (a) untreated control group, (b–d) rats treated with EG for (b) 7, (c) 21, or (d) 42 days. Right panels: (e) E-treated control group, (f–h) rats treated with EG+E for (f) 7, (g) 21, or (h) 42 days. Reduced from × 400. Kidney International 2006 70, 699-710DOI: (10.1038/sj.ki.5001651) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 5 Representative micrographs of renal sections stained for μGST. Left panels: (a) untreated control group, (b–d) rats treated with EG for (b) 7, (c) 21, or (d) 42 days. Right panels: (e) E-treated control group, (f–h) rats treated with EG+E for (f) 7, (g) 21, or (h) 42 days. Reduced from × 400. Kidney International 2006 70, 699-710DOI: (10.1038/sj.ki.5001651) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 6 Representative micrographs of renal tubules stained for nuclear PCNA. PCNA-positive cells are indicated by arrows. Left panels: (a) untreated control group, (b–d) rats treated with EG for (b) 7, (c) 21, or (d) 42 days. Right panels: (e) E-treated control group, (f–h) rats treated with EG+E for (f) 7, (g) 21, or (h) 42 days. (i) Statistical result for cell counting. *P<0.05 compared to the corresponding control group. *P<0.05 compared to the EG group at the same time point. Reduced from × 400. Kidney International 2006 70, 699-710DOI: (10.1038/sj.ki.5001651) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 7 Apoptotic cells in the kidneys evaluated using TUNEL staining. Left panels: (a) untreated control group, (b–d) rats treated with EG for (b) 7, (c) 21, or (d) 42 days. Right panels: (e) E-treated control group, (f–h) rats treated with EG+E for (f) 7, (g) 21, or (h) 42 days. (i) Statistical result for cell counting. *P<0.05 compared to the corresponding control group. Reduced from × 400. Kidney International 2006 70, 699-710DOI: (10.1038/sj.ki.5001651) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 8 Effect of vitamin E on tubular cell death and proliferation in the EG-treated kidneys. Levels of (a) αGST, (b) μGST, and (c) PCNA, and (d) cytochrome c release in the kidneys. The representative blots shown were obtained from two different rat kidneys. Since there was no significant difference between control groups, one group of controls is shown. The histogram shows the relative density of the protein of interest to β-actin for six animals in each group. EG, ethylene glycol; E, vitamin E treatment; 7, 21, and 42 represent days of administration; P, positive control. *P<0.05 compared to the age-matched corresponding control group. Kidney International 2006 70, 699-710DOI: (10.1038/sj.ki.5001651) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 9 Effect of vitamin E on the expressions of anticrystallization molecules in the EG-treated kidneys. Levels of osteopontin and Tamm-Horsfall protein in the (a) renal medulla and (b) urine at day 42. The representative blots show the band for the protein of interest in samples from three different animals. The bar graphs show the changes in OPN or THP levels in renal tissues or urine for six animals. *P<0.05 compared to the corresponding control group. †P<0.05 compared to the EG group. Kidney International 2006 70, 699-710DOI: (10.1038/sj.ki.5001651) Copyright © 2006 International Society of Nephrology Terms and Conditions